CN103983714A - Method for measuring contents of coumarin and safrole in tobacco products - Google Patents
Method for measuring contents of coumarin and safrole in tobacco products Download PDFInfo
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- CN103983714A CN103983714A CN201410245020.5A CN201410245020A CN103983714A CN 103983714 A CN103983714 A CN 103983714A CN 201410245020 A CN201410245020 A CN 201410245020A CN 103983714 A CN103983714 A CN 103983714A
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Abstract
The invention provides a method for measuring the contents of coumarin and safrole in tobacco products. The method comprises the steps of carrying out ultrasonic extraction, filtering with an organic filter membrane and microextraction on the tobacco products to obtain a tobacco product solution to be measured; and performing gas chromatography-mass spectrometry detection on the tobacco product solution to be measured, thus obtaining the contents of coumarin and safrole in the tobacco products according to an acquired chromatogram of the tobacco product and preset standard curves of coumarin and safrole, so that the effect of simultaneously measuring coumarin and safrole in the tobacco products is achieved. The method can rapidly and accurately measure the contents of coumarin and safrole in the tobacco products, reduces the matrix effects and improves the separation degree of coumarin and safrole by adopting the microextraction technology; and the method has simplicity and convenience in operation.
Description
Technical field
The present invention relates to tobacco product physico-chemical examination technique field, relate in particular to a kind of method of measuring cumarin and safrole content in tobacco product.
Background technology
Cumarin (coumarin), has another name called for adjacent hydroxyl cinnamic acid lactone and 1,2-benzopyrone, is the lactone compound extensively existing at occurring in nature, in Rutaceae and samphire, at most, also in other section plant, exists.Studies have reported that, cumarin is toxin precursor, can be converted into bicoumarin, and bicoumarin has blood coagulation resisting function, can cause animal capillary hemorrhage, finally makes pluck organ impaired.Safrole (safrole) is also referred to as safrole, 1-ally-3,4-methy-lene dioxy benzene, safrole, mainly be present in the essential oil of the cinnamons such as heavy water camphor tree, hard leaf camphor tree, also being a kind of important source material of the spices such as synthesizing piperonal, vanillic aldehyde and piperonyl butoxide, is also a kind of natural essence.But studies show that, safrole forms safrole-DNA plus compound in hepatic tissue, to human body toxic side effect, easily modificator gene sudden change and hepatic injury, or even the strong carcinogen of digestion, hematological system, safrole is that countries in the world all ban use of.
At present the detection of cumarin and safrole is mainly concentrated in cosmetics, essence and flavoring agent and traditional Chinese medicine sample, detection method has the pre-treating methods such as while distillation extraction, headspace extraction technology, supercritical fluid technique and solid phase micro-extraction technique and gas chromatography (GC), Gas Chromatography/Mass Spectrometric Method (GC-MS), gas chromatography-tandem mass spectrum (GC-MS/MS) and high performance liquid chromatography (HPLC) etc. to analyze Instrument crosslinking analysis.The shortcomings such as that but these sample-pretreating methods exist is consuming time, consumption power, consumption solvent.
Due in essence, spices likely natural plant essential oils bring the compositions such as cumarin, safrole into, can, by the essence, the spices that add in pipe tobacco, more or less be present in tobacco product.Therefore,, in order to control the security of tobacco product well, it is very necessary setting up the method that simultaneously detects fast cumarin and safrole.
Summary of the invention
In view of this, the object of the invention is to a kind of method of measuring cumarin and safrole content in tobacco product, method provided by the invention can be fast, accurately, reduce matrix effect, improved the degree of separation of cumarin and safrole, realize the mensuration to cumarin and safrole simultaneously.
The invention provides a kind of method of measuring cumarin and safrole content in tobacco product, comprise the following steps:
Tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid;
Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate;
Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured;
Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco product;
According to the chromatogram of described tobacco product and predetermined cumarin and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.
Preferably, the extraction solvent of described ultrasonic extraction is methylene chloride;
The time of described ultrasonic extraction is 15min～25min.
Preferably, the volume ratio of the quality of described tobacco product testing sample and the extraction solvent of ultrasonic extraction is 0.5g:(10～20) mL.
Preferably, the aperture of described organic filter membrane is 0.45 μ m.
Preferably, the extraction solvent of described micro-extraction is methylene chloride.
Preferably, the time of described micro-extraction is 3min～5min.
Preferably, the temperature of described micro-extraction is 140 ℃～220 ℃.
Preferably, in described gas chromatography-mass spectrum detection, the condition of gas chromatography is:
Gas chromatographic sample introduction mouth temperature is 270 ℃～290 ℃;
Gas chromatography flow rate of carrier gas is 1.00mL/min;
The heating schedule of gas chromatography is gradient increased temperature.
Preferably, described gradient increased temperature is:
In initial temperature, keep the very first time, with the first heating rate, be warming up to the first temperature, described initial temperature is 90 ℃～110 ℃, and the described very first time is 2min～4min, described the first heating rate is 4 ℃/min～8 ℃/min, and described the first temperature is 150 ℃～170 ℃;
With the second heating rate, be warming up to the second temperature again, keeping the second time, described the second heating rate is 18 ℃/min～25 ℃/min, and described the second temperature is 270 ℃～290 ℃, and described the second time is 3min～7min.
Preferably, in described gas chromatography-mass spectrum detection, mass spectrographic condition is:
Mass spectrum transmission line temperature is 275 ℃～285 ℃;
Mass ion source temperature is 225 ℃～235 ℃;
Mass spectrum level Four bar temperature is 145 ℃～155 ℃;
The mass spectrum solvent delay time is 3min～5min;
Mass spectrum ionization mode is EI source;
Mass spectrum ionizing energy is 65eV～75eV;
Scanning of the mass spectrum mode is for selecting ion monitoring pattern.
The invention provides a kind of method of measuring cumarin and safrole content in tobacco product, comprise the following steps: tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid; Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate; Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured; Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco sample; According to the chromatogram of described tobacco sample and predetermined cumarin typical curve and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.Method provided by the invention is carried out ultrasonic extraction, organic membrane filtration and micro-extraction by tobacco product, obtains the solution to be measured of tobacco product; The solution to be measured of described tobacco product is carried out to gas chromatography-mass spectrum detection, when having realized the cumarin in tobacco product and safrole, measure.Method provided by the invention can realize the detection to cumarin in tobacco product and safrole content quickly and accurately, by micro-extraction technique, has reduced matrix effect, improved cumarin and safrole degree of separation, and method is easy and simple to handle.
Experimental result shows, utilizes the present invention can measure accurately cumarin and safrole content in tobacco product, and the method recovery is 85%～97%, and average relative standard deviation (RSD) is less than 6.5, illustrates that the inventive method recovery is high, reproducible; Sample preparation is easy and simple to handle, sample, on the multi-functional micro-extraction instrument of ME-101, can a step complete extraction, the purification, concentrated of sample, and greatly eliminate sample substrate the chromatogram of object is disturbed, the chromatographic resolution degree of object is better, is conducive to accurately qualitative, quantitative; The multi-functional micro-extraction instrument of ME-101 extraction time is 4min only, and analysis time is short, realizes the robotization of sample pre-treatments, can process tobacco product sample in enormous quantities, ensures quality and the security of tobacco product.
Accompanying drawing explanation
Fig. 1 is the cumarin that obtains of the embodiment of the present invention 1 and the stratographic analysis figure of safrole standard items;
Fig. 2 is the histogram of the extraction temperature that obtains of the embodiment of the present invention 2 on cumarin and the impact of safrole extraction efficiency;
Fig. 3 is the histogram of the extraction time that obtains of the embodiment of the present invention 3 on cumarin and the impact of safrole extraction efficiency;
Fig. 4 is that the tobacco product that the embodiment of the present invention 9 obtains adds target stratographic analysis figure;
Fig. 5 is other tobacco product matrix recovery of standard addition histogram of variable concentrations level that the embodiment of the present invention 9 obtains.
Embodiment
The invention provides a kind of method of measuring cumarin and safrole content in tobacco product, comprise the following steps:
Tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid;
Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate;
Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured;
Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco sample;
According to the chromatogram of described tobacco sample and predetermined cumarin typical curve and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.
Method provided by the invention, by tobacco product ultrasonic extraction, organic membrane filtration and micro-extraction, extracts cumarin and safrole in tobacco product simultaneously, obtains the solution to be measured of tobacco product; Again the solution to be measured of described tobacco product is carried out to gas chromatography-mass spectrum detection, when having realized cumarin and safrole, measure.The present invention has set up a kind of method that while fast, accurately detects cumarin and safrole in tobacco product, by micro-extraction technique, has reduced matrix effect, improved degree of separation, and method is easy and simple to handle.
The present invention carries out ultrasonic extraction by tobacco product testing sample, obtains ultrasonic extraction liquid.The present invention does not have special restriction to the kind of described tobacco product, can be various pipe tobacco or the cigarette of smoking well known to those skilled in the art.In the present invention, described tobacco product testing sample is preferably solid-state tobacco product, and the granularity of described solid-state tobacco product testing sample is preferably-200 orders, and in the present invention-200 orders refer to the screen underflow of 200 mesh sieves.The present invention is preferably dried tobacco product, pulverize and sieves, and obtains tobacco product testing sample.The present invention does not have special restriction to described dry method, adopts well known to those skilled in the art tobacco to be carried out to dry technical scheme; In the present invention, the described dry freeze drying that is preferably; In the present invention, the water percentage of described dried tobacco product preferably≤6%, more preferably≤5%.The present invention does not have special restriction to the method for described pulverizing, adopts the technical scheme of pulverizing well known to those skilled in the art, as adopted the mode of underhand polish, can adopt the mode of mechanical disintegration yet.In the present invention, described in, sieve and preferably adopt 200 mesh sieves.
Obtain after tobacco product testing sample, the present invention carries out ultrasonic extraction by described tobacco product testing sample, obtains ultrasonic extraction liquid.In the present invention, the extraction solvent of described ultrasonic extraction is preferably methylene chloride; The quality of described tobacco product testing sample and the volume ratio of methylene chloride are preferably 0.5g:(10～20) mL, more preferably 0.5g:15mL; The time of described ultrasonic extraction is preferably 15min～25min, more preferably 20min.
Complete described ultrasonic extraction, obtain after ultrasonic extraction liquid, the present invention through organic membrane filtration, obtains filtrate by described ultrasonic extraction liquid.The present invention does not have special restriction to the material of described organic filter membrane, adopts organic filter membrane well known to those skilled in the art; In the present invention, the aperture of described organic filter membrane is preferably 0.45 μ m.The present invention preferably by ultrasonic extraction liquid through organic membrane filtration, the concentrated filtrate that obtains 90 μ L～110 μ L, more preferably concentratedly obtains 100 μ L filtrates.
Obtain after filtrate, the present invention adopts gas flow purging micro-syringe abstraction technique, and described filtrate is carried out to micro-extraction, obtains tobacco product solution to be measured.It is that the micro-extraction instrument of ME-101 carries out micro-extraction to described filtrate that the present invention preferably adopts model, preferably 15 μ L～25 μ L filtrates is placed in to the sample cell of described micro-extraction instrument, with dottle pin by the sealing of sample cell mouth, more preferably 20 μ L; Then the micro-syringe that by volume is 100 μ L is vertically inserted in sample cell through dottle pin through condensing unit, adds extraction solvent to carry out micro-extraction in described micro-syringe.In the present invention, the extraction solvent that described micro-extraction adopts is preferably methylene chloride; In the present invention, the time of described micro-extraction is preferably 3min～5min, more preferably 4min; The temperature of described micro-extraction is preferably 140 ℃～220 ℃, more preferably 150 ℃～200 ℃, most preferably is 160 ℃.
Complete after described micro-extraction, for the ease of the content of cumarin and safrole in detection tobacco sample, the present invention is preferably settled to 100 μ L by the micro-extraction liquid obtaining, and obtains tobacco sample solution to be measured.
Obtain after tobacco sample solution to be measured, the present invention carries out gas chromatography-mass spectrum detection by described tobacco sample solution to be measured, obtains the chromatogram of tobacco sample.The present invention detects to described gas chromatography-mass spectrum the instrument adopting does not have special restriction, adopts gas chromatograph-mass spectrometer (GCMS) well known to those skilled in the art.In the present invention, during described gas chromatography-mass spectrum detects, the condition of gas chromatography is preferably:
The sample size of described gas chromatography is preferably 1 μ L～3 μ L, more preferably 2 μ L; The chromatographic column of described gas chromatography is DB-5MS quartz capillary column, and described chromatographic column is preferably dimensioned to be 30m * 0.25mm * 0.25 μ m; The temperature of described gas chromatographic sample introduction mouth is preferably 270 ℃～290 ℃, more preferably 275 ℃～285 ℃, most preferably is 280 ℃; The carrier gas of described gas chromatography is preferably high-purity helium, and the purity of described high-purity helium is preferably 99.999%; The mobile constant current mode that is preferably of described carrier gas, the flow velocity of described carrier gas is preferably 1.00mL/min; Described gas chromatographic detection is not shunted; The heating schedule of described gas chromatography is preferably gradient increased temperature; Described gradient increased temperature is preferably:
In initial temperature, keep the very first time, with the first heating rate, be warming up to the first temperature, described initial temperature is preferably 90 ℃～110 ℃, more preferably 95 ℃～105 ℃, most preferably is 100 ℃; The described very first time is preferably 2min～4min, more preferably 3min; Described the first heating rate is preferably 4 ℃/min～8 ℃/min, more preferably 6 ℃/min; Described the first temperature is preferably 150 ℃～170 ℃, more preferably 155 ℃～165 ℃, most preferably is 160 ℃;
With the second heating rate, be warming up to the second temperature again, keep the second time, described the second heating rate to be preferably 18 ℃/min～25 ℃/min, more preferably 20 ℃/min; Described the second temperature is preferably 270 ℃～290 ℃, more preferably 275 ℃～285 ℃, most preferably is 280 ℃; Described the second time is preferably 3min～7min, and more preferably 4min～6min, most preferably is 5min.
In the present invention, in described gas chromatography-mass spectrum detection, mass spectrographic condition is preferably:
Described mass spectrum transmission line temperature is 275 ℃～285 ℃, more preferably 280 ℃;
Described mass ion source temperature is 225 ℃～235 ℃, more preferably 230 ℃;
Described mass spectrum level Four bar temperature is 145 ℃～155 ℃, more preferably 150 ℃;
The described mass spectrum solvent delay time is 3min～5min, more preferably 4min;
Described mass spectrum ionization mode is EI source;
Described mass spectrum ionizing energy is 65eV～75eV, more preferably 70eV;
Described scanning of the mass spectrum mode is for selecting ion monitoring pattern.
After completing described gas chromatography-mass spectrum and detecting, the present invention is according to the chromatogram of the tobacco product obtaining and predetermined cumarin and safrole typical curve, obtains the content of cumarin and safrole in tobacco product.
In the present invention, the typical curve of described cumarin and safrole preferably obtains in accordance with the following methods:
The standard operation solution of preparation series concentration, described standard operation solution comprises cumarin and safrole;
The standard operation solution of described series concentration is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of standard operation liquid;
The peak area of cumarin in the chromatogram of described standard operation liquid, safrole of take is respectively ordinate, and the concentration that cumarin and safrole are corresponding is that horizontal ordinate carries out regretional analysis, obtains the typical curve of cumarin and safrole.
The present invention does not have special restriction to the method for preparation standard operation liquid, adopts the method for preparing standard solution well known to those skilled in the art.Concrete, the present invention can first prepare standard reserving solution, then standard reserving solution is diluted to desired concn.The present invention preferably prepares two-stage standard reserving solution, is respectively primary standard storing solution, called after standard reserving solution I, and secondary standard storing solution, called after standard reserving solution II.In the present invention, the solvent in described standard reserving solution I is preferably methylene chloride; In described standard reserving solution I, the mass concentration of cumarin is preferably 500.0mgL
-1, in described standard reserving solution I, the mass concentration of safrole is preferably 500.0mgL
-1; Solvent in described standard reserving solution II is preferably methylene chloride; In described standard reserving solution II, the mass concentration of cumarin is preferably 5mgL
-1, in described standard reserving solution II, the mass concentration of safrole is preferably 5mgL
-1.
The present invention is placed in respectively volumetric flask by the standard reserving solution I of the standard reserving solution II of serial volume and certain volume, obtains other standard solution of series concentration level; With methylene chloride, be diluted to scale, obtain the standard operation liquid of series concentration.In an embodiment of the present invention, the volume of described standard reserving solution II can be 0.1mL, 0.2mL, 1mL and 2mL; The volume of described standard reserving solution I can be 0.1mL; Those skilled in the art can be as required, the standard operation liquid of preparation variable concentrations, and the present invention does not have special restriction to this.
Obtain after the standard operation liquid of series concentration, the technical scheme that the present invention preferably adopts the gas chromatography-mass spectrum described in technique scheme to detect, carries out gas chromatography-mass spectrum detection to described standard operation liquid, obtains the chromatogram of standard operation liquid.
Obtain after the chromatogram of standard operation liquid, the present invention carries out integration to cumarin wherein and the chromatographic peak of safrole, obtains the peak area of the chromatographic peak of cumarin, safrole.The present invention be take the peak area of cumarin and safrole as ordinate carries out regretional analysis, obtains the typical curve of cumarin and safrole.
The chromatogram of the tobacco product that the present invention obtains according to technique scheme, obtains the wherein peak area of cumarin and safrole; Again according to the typical curve of described cumarin and safrole, calculate the concentration of cumarin and safrole in tobacco product solution to be measured; Again according to the volume of tobacco product solution to be measured, calculate the quality of cumarin and safrole in tobacco product solution to be measured; Again according to the quality of tobacco product testing sample, calculate the content of cumarin and safrole in tobacco product.
Method provided by the invention has also been investigated detectability, the recovery and the precision of the method, and described detectability can specifically obtain by the following method:
According to cumarin and safrole typical curve, obtain the least concentration of cumarin and safrole;
The standard operation liquid of cumarin and safrole least concentration is carried out to ten parallel laboratory tests, and the standard deviation of 3 times calculating is that detection limit, the standard deviation of 10 times are quantitative detection limit.Result demonstration, method provided by the invention is limited to 0.021 μ g/g to detecting of cumarin, and detecting of safrole is limited to 0.015 μ g/g; Cumarin be quantitatively limited to 0.07 μ g/g, safrole be quantitatively limited to 0.05 μ g/g.
In the present invention, the described recovery can obtain by the following method:
The standard operation liquid that adds respectively series concentration in blank tobacco product, carries out micro-extraction according to the method for micro-extraction described in technique scheme, obtains solution to be measured;
Described solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the wherein content of cumarin and safrole;
Every group of parallel carrying out three times of experiment, is averaged result.
Experimental result shows, method provided by the invention, and the recovery of standard addition of cumarin is 80%～87%, and the recovery of standard addition 83%～97% of safrole, RSD value is less than 10.57%.This explanation, the method recovery is high, reproducible, when being applicable in tobacco product cumarin and safrole, detects.
The invention provides a kind of method of measuring cumarin and safrole content in tobacco product, comprise the following steps: tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid; Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate; Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured; Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco sample; According to the chromatogram of described tobacco sample and predetermined cumarin typical curve and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.Method provided by the invention is carried out ultrasonic extraction, organic membrane filtration and micro-extraction by tobacco product, obtains the solution to be measured of tobacco product; The solution to be measured of described tobacco product is carried out to gas chromatography-mass spectrum detection, when having realized the cumarin in tobacco product and safrole, measure.Method provided by the invention can realize the detection to cumarin in tobacco product and safrole content quickly and accurately, by micro-extraction technique, has reduced matrix effect, make cumarin and safrole have good degree of separation, and method is easy and simple to handle.
Below in conjunction with embodiment, the method for cumarin and safrole content in mensuration tobacco product provided by the invention is described in detail, but they can not be interpreted as to limiting the scope of the present invention.
In the following embodiments, tobacco product sample is selected from cigarette enterprise; Cumarin is purchased from German Dr.Ehrenstorfer; Safrole standard items are purchased from U.S. AccuStandard; Organic filter membrane is purchased from U.S. PALL; ; Methylene chloride is chromatographically pure, purchased from Canadian Clayton Corp.; The multi-functional micro-extraction Yi You of ME-101 Yanbian University provides; GC-MS2010 is purchased from Japanese Shimadzu company; Electronic analytical balance is purchased from Switzerland's plum Teller precision balance.
Embodiment 1
Take respectively the cumarin of 0.0500g and 0.0500g safrole standard items in 100mL volumetric flask, with methylene chloride dilution, be settled to scale and shake up, obtain 500.0mgL
-1mixed standard solution is standard reserving solution I.Then with methylene chloride, being diluted to concentration is 5mgL again
-1, shake up, be placed in stored under refrigeration, standby, obtain standard reserving solution II.
The hybrid standard storing solution II and the 0.1mL hybrid standard storing solution I that pipette respectively 0.1mL, 0.2mL, 1mL, 2mL, being placed in respectively 5 volumes is 10mL volumetric flask, obtains other standard operation solution of 5 concentration levels, with methylene chloride, is settled to scale, shakes up.The concentration of 5 standard solution is followed successively by 0.05mgL
-1, 0.1mgL
-1, 0.5mgL
-1, 1mgL
-1, 5mgL
-1.
To obtain standard solution and carry out Gas Chromatography-Mass Spectrometry, wherein the condition of gas Chromatographic Determination is:
Chromatographic column, DB-5MS quartz capillary column (30m * 0.25mm * 0.25 μ m); Injector temperature: 280 ℃; Carrier gas: high-purity helium, purity is 99.999%; The constant current mode of flow velocity: 1.00mL/min; Split ratio: do not shunt; Heating schedule: initial temperature is 100 ℃, keeps 3min, is increased to 160 ℃, then is increased to 280 ℃ with 20 ℃/min with 6 ℃/min, keeps 5min;
Mass spectrum condition is: transmission line temperature: 280 ℃; Ion source temperature: 230 ℃; Level Four bar temperature: 150 ℃; Solvent delay: 4min; Ionization mode: EI source; Ionizing energy: 70eV; Scan mode: select ion monitoring pattern (SIM).
Detection obtains the gas chromatogram of standard solution, and as shown in Figure 1, Fig. 1 is the cumarin that obtains of the embodiment of the present invention 1 and the stratographic analysis figure of safrole standard items to result; According to the stratographic analysis figure of the standard solution of series concentration, to the peak area of cumarin, safrole wherein, be ordinate, with the respective concentration of cumarin and safrole, carry out regretional analysis, obtain the typical curve of tonka-bean and safrole.
Embodiment 2
Get tobacco product sample, after freeze drying, make moisture keep 5% left and right.By sample pulverizing, 200 mesh sieves that sieve, sample powder under the sieve after sieving is put into brown sample bottle refrigeration, to be detected;
Get the powder of 0.5g tobacco product sample, with 15mL methylene chloride ultrasonic extraction 20min, extract, through the organic filter membrane of 0.45 μ m, is concentrated into 100 μ L afterwards.Get the extract of 20 μ L, be filled in the sample cell of ME-101 micro-extraction instrument, with dottle pin sealed sample Chi Kou, 100 μ L micro-syringe are vertically inserted in sample cell through condensing unit, to appropriate location under dottle pin, in micro-syringe, add 10 μ L extraction solvents to start extraction, extraction time is 4min.The multi-functional micro-extraction instrument parameter of ME-101 is:
Gas flow purging micro-syringe abstraction technique extraction conditions is for as follows: nitrogen flow rate is 2mL/min, and extraction temperature is respectively 140 ℃, 160 ℃, 180 ℃, 220 ℃, 260 ℃, 300 ℃, and extraction time is 4min, and condensing temperature is-4 ℃.
As shown in Figure 2, Fig. 2 is the histogram of the extraction temperature that obtains of the embodiment of the present invention 2 on cumarin and the impact of safrole extraction efficiency to result, and result shows, under 140 ℃ of extraction temperature, the recovery of cumarin and safrole is respectively 70%, 72%; Extraction temperature recovery of cumarin and safrole under 160 ℃～300 ℃ conditions be respectively 82%～99% and 84%～98%, RSD value be less than 11.5%, meet the requirement of the recovery.
Embodiment 3
Adopt the method for embodiment 2, the impact of test extraction time on cumarin and safrole extraction efficiency, different, in the present embodiment, extraction temperature is 160 ℃, and extraction time is respectively 3min, 4min, 5min, and each tests parallel doing three times.
Result as shown in Figure 3, Fig. 3 is the histogram of the extraction time that obtains of the embodiment of the present invention 3 on cumarin and the impact of safrole extraction efficiency, and result shows, when extraction time is 3min, cumarin and safrole average recovery rate be 69% and 75%, RSD value be less than 15%; And extraction time is while being 4～5min, the average recovery rate of object is 92%～114%, and relative standard deviation is less than 9.91%, and the recovery and precision all meet analyzes requirement.
Embodiment 4
Get tobacco product sample, after freeze drying, by sample pulverizing, 200 mesh sieves that sieve, the sample powder after sieving is put into brown sample bottle refrigeration, to be detected;
Get the powder of 0.5g tobacco product sample, with 15mL methylene chloride ultrasonic extraction 20min, extract, through the organic filter membrane of 0.45 μ m, is concentrated into 100 μ L afterwards.Get the extract of 20 μ L, be filled in the sample cell of ME-101 micro-extraction instrument, with dottle pin sealed sample Chi Kou, 100 μ L micro-syringe are vertically inserted in sample cell through condensing unit, to appropriate location under dottle pin, in micro-syringe, add 10 μ L extraction solvents to start extraction, extraction time is 4min.The multi-functional micro-extraction instrument parameter of ME-101 is:
Gas flow purging micro-syringe abstraction technique extraction conditions is for as follows: nitrogen flow rate is 2mL/min, and extraction temperature is 160 ℃, and extraction time is 4min, and condensing temperature is-4 ℃.
After extraction finishes, final constant volume is 100 μ L, gets 2 μ L and enters gas chromatography mass spectrometer analysis, and gas chromatography-mass spectrum condition is:
Chromatographic column, DB-5MS quartz capillary column (30m * 0.25mm * 0.25 μ m); Injector temperature: 280 ℃; Carrier gas: high-purity helium, purity is 99.999%; The constant current mode of flow velocity: 1.00mL/min; Split ratio: do not shunt; Heating schedule: initial temperature is 100 ℃, keeps 3min, is increased to 160 ℃, then is increased to 280 ℃ with 20 ℃/min with 6 ℃/min, keeps 5min;
Mass spectrum condition is: transmission line temperature: 280 ℃; Ion source temperature: 230 ℃ of level Four bar temperature: 150 ℃; Solvent delay: 4min; Ionization mode: EI source; Ionizing energy: 70eV; Scan mode: select ion monitoring pattern (SIM).
Detection obtains the ion of Mass Spectrometer Method: qualitative, the quota ion of cumarin are respectively m/z146 and m/z118, and qualitative, the quota ion of safrole are respectively m/z131 and m/z162;
According to the peak area of cumarin and safrole chromatographic peak in the stratographic analysis figure obtaining, and the cumarin that obtains of embodiment 1 and the typical curve of safrole, calculate the content of cumarin and safrole in tobacco product, result is as shown in table 1, the test result that table 1 obtains for the embodiment of the present invention 4～8.
Embodiment 5～8
Adopt the technical scheme described in embodiment 4, other 5 kinds of samples from the tobacco product of kinds of cigarettes enterprise are detected, obtain the wherein content of cumarin and safrole, result is as shown in table 1, the test result that table 1 obtains for the embodiment of the present invention 4～8.
The test result that table 1 embodiment of the present invention 4～8 obtains
| Embodiment | Safrole (μ gg -1) | Cumarin (μ gg -1) |
| 4 | ND | 0.07 |
| 5 | ND | ND |
| 6 | ND | 0.15 |
| 7 | ND | ND |
| 8 | ND | 0.10 |
Note: ND is not for detecting.
As can be seen from Table 1, all do not detect safrole in the sample of tobacco product, in a small amount of sample, cumarin detected, concentration range is ND～0.15 μ gg
-1, RSD is 3.5%～7.6%.
Embodiment 9
In blank tobacco product sample, add respectively 0.1 μ gg
-1, 0.5 μ gg
-1, 5.0 μ gg
-1with 50.0 μ gg
-1cumarin and the safrole of Different adding amount level, adopt micro-extraction and gas chromatography-mass spectrum detection method described in embodiment 4 to detect, every group of parallel carrying out three times of experiment.
Result as shown in Figure 4 and Figure 5, Fig. 4 is that the tobacco product that the embodiment of the present invention 9 obtains adds target stratographic analysis figure, Fig. 5 is other tobacco product matrix recovery of standard addition histogram of variable concentrations level that the embodiment of the present invention 9 obtains, result shows, the recovery of standard addition of cumarin is 80%～87%, the recovery of standard addition of safrole is 83%～97%, RSD value is less than 10.57%, this explanation, the method recovery provided by the invention is high, reproducible, when being applicable in tobacco product cumarin and safrole, detect.
As seen from the above embodiment, the invention provides a kind of method of measuring cumarin and safrole content in tobacco product, comprise the following steps: tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid; Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate; Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured; Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco sample; According to the chromatogram of described tobacco sample and predetermined cumarin typical curve and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.Method provided by the invention is carried out ultrasonic extraction, organic membrane filtration and micro-extraction by tobacco product, obtains the solution to be measured of tobacco product; The solution to be measured of described tobacco product is carried out to gas chromatography-mass spectrum detection, when having realized the cumarin in tobacco product and safrole, measure.Method provided by the invention can realize the detection to cumarin in tobacco product and safrole content quickly and accurately, by micro-extraction technique, has reduced matrix effect, have degree of separation good, and method is easy and simple to handle.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a method of measuring cumarin and safrole content in tobacco product, comprises the following steps:
Tobacco product testing sample is carried out to ultrasonic extraction, obtain ultrasonic extraction liquid;
Described ultrasonic extraction liquid, through organic membrane filtration, is obtained to filtrate;
Adopt gas flow purging micro-syringe abstraction technique, described filtrate is carried out to micro-extraction, obtain tobacco product solution to be measured;
Described tobacco product solution to be measured is carried out to gas chromatography-mass spectrum detection, obtain the chromatogram of tobacco product;
According to the chromatogram of described tobacco product and predetermined cumarin and safrole typical curve, obtain the content of cumarin and safrole in tobacco product.
2. method according to claim 1, is characterized in that, the extraction solvent of described ultrasonic extraction is methylene chloride;
The time of described ultrasonic extraction is 15min～25min.
3. method according to claim 1 and 2, is characterized in that, the volume ratio of the quality of described tobacco product testing sample and the extraction solvent of ultrasonic extraction is 0.5g:(10～20) mL.
4. method according to claim 1, is characterized in that, the aperture of described organic filter membrane is 0.45 μ m.
5. method according to claim 1, is characterized in that, the extraction solvent of described micro-extraction is methylene chloride.
6. method according to claim 1 or 5, is characterized in that, the time of described micro-extraction is 3min～5min.
7. method according to claim 1 or 5, is characterized in that, the temperature of described micro-extraction is 140 ℃～220 ℃.
8. method according to claim 1, is characterized in that, during described gas chromatography-mass spectrum detects, the condition of gas chromatography is:
Gas chromatographic sample introduction mouth temperature is 270 ℃～290 ℃;
Gas chromatography flow rate of carrier gas is 1.00mL/min;
The heating schedule of gas chromatography is gradient increased temperature.
9. method according to claim 8, is characterized in that, described gradient increased temperature is:
In initial temperature, keep the very first time, with the first heating rate, be warming up to the first temperature, described initial temperature is 90 ℃～110 ℃, and the described very first time is 2min～4min, described the first heating rate is 4 ℃/min～8 ℃/min, and described the first temperature is 150 ℃～170 ℃;
With the second heating rate, be warming up to the second temperature again, keeping the second time, described the second heating rate is 18 ℃/min～25 ℃/min, and described the second temperature is 270 ℃～290 ℃, and described the second time is 3min～7min.
10. method according to claim 1, is characterized in that, during described gas chromatography-mass spectrum detects, mass spectrographic condition is:
Mass spectrum transmission line temperature is 275 ℃～285 ℃;
Mass ion source temperature is 225 ℃～235 ℃;
Mass spectrum level Four bar temperature is 145 ℃～155 ℃;
The mass spectrum solvent delay time is 3min～5min;
Mass spectrum ionization mode is EI source;
Mass spectrum ionizing energy is 65eV～75eV;
Scanning of the mass spectrum mode is for selecting ion monitoring pattern.
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| CN107561184A (en) * | 2017-09-05 | 2018-01-09 | 中国检验检疫科学研究院 | A kind of method of coumarin kind compound in detection toy for children |
| CN108845067A (en) * | 2018-04-28 | 2018-11-20 | 江苏中烟工业有限责任公司 | A kind of purge and trap method of flavor component in tobacco and tobacco product |
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| CN106841451A (en) * | 2017-02-24 | 2017-06-13 | 中国烟草总公司郑州烟草研究院 | The assay method of cumarin and its metabolin in mouse blood based on Ultra Performance Liquid Chromatography track trap high resolution mass spectrum |
| CN107561184A (en) * | 2017-09-05 | 2018-01-09 | 中国检验检疫科学研究院 | A kind of method of coumarin kind compound in detection toy for children |
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| CN108845067A (en) * | 2018-04-28 | 2018-11-20 | 江苏中烟工业有限责任公司 | A kind of purge and trap method of flavor component in tobacco and tobacco product |
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